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A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

Cahn, Jackson K. B. and Werlang, Caroline A. and Baumschlager, Armin and Brinkmann-Chen, Sabine and Mayo, Stephen L. and Arnold, Frances H. (2017) A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases. ACS Synthetic Biology, 6 (2). pp. 326-333. ISSN 2161-5063. PMCID PMC8611728. doi:10.1021/acssynbio.6b00188.

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The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal–Structural Analysis and Library Design (CSR-SALAD).

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Cahn, Jackson K. B.0000-0001-8849-4516
Werlang, Caroline A.0000-0001-7554-8431
Baumschlager, Armin0000-0002-2546-9038
Brinkmann-Chen, Sabine0000-0002-5419-4192
Mayo, Stephen L.0000-0002-9785-5018
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2017 American Chemical Society. Received: July 7, 2016; Published: September 20, 2016. This work was supported by the Gordon and Betty Moore Foundation through Grant No. GBMF2809 to the Caltech Programmable Molecular Technology Initiative and by American Recovery and Reinvestment Act (ARRA) funds through the National Institutes of Health Shared Instrumentation Grant Program, Grant No. S10RR027203, to F.H.A. J.K.B.C. acknowledges the support of the Resnick Sustainability Institute (Caltech). The authors thank Ruchi Jahagirdar and Lisa Mears for experimental assistance and Tilman Flock for providing the list of nonredundant PDBs used for CSR-SALAD testing and validation. They also thank numerous former and current members of the Arnold and Mayo laboratories for invaluable suggestions and discussions. Accession Codes: PDB: 4XDY, PDB: 4TSK, PDB: 3DOJ, PDB: 1PIW, PDB: 1K8C, PDB: 1VHD, PDB: 3PEF Author Contributions: This project was conceived by J.K.B.C., S.B-C., S.L.M., and F.H.A. J.K.B.C. designed and programmed CSR-SALAD, which was implemented online by C.A.W. J.K.B.C. performed the validation experiments with assistance from A.B., and produced the figures and tables. J.K.B.C., S.B-C., and F.H.A. wrote the manuscript with input from all authors. The authors declare no competing financial interest.
Group:Resnick Sustainability Institute
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF2809
Resnick Sustainability InstituteUNSPECIFIED
Subject Keywords:cofactor specificity, oxidoreductases, protein engineering, library design, semirational engineering
Issue or Number:2
PubMed Central ID:PMC8611728
Record Number:CaltechAUTHORS:20161012-112258970
Persistent URL:
Official Citation:A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases Jackson K. B. Cahn, Caroline A. Werlang, Armin Baumschlager, Sabine Brinkmann-Chen, Stephen L. Mayo, and Frances H. Arnold ACS Synthetic Biology 2017 6 (2), 326-333 DOI: 10.1021/acssynbio.6b00188
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:71018
Deposited By: Ruth Sustaita
Deposited On:12 Oct 2016 21:57
Last Modified:14 Apr 2022 17:21

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